It is well established that diabetes leads to a marked increased risk for the development of heart failure independent of other risk factors; however, there is no consensus as to the mechanisms involved or the most appropriate treatment strategies. The O-linked attachment of ss-N-acetyl- glucosamine (O-GlcNAc) to serine and threonine residues is a highly dynamic post-translational modification of nuclear and cytoplasmic proteins. This novel, metabolically regulated signaling pathway is emerging as a key regulator of critical biological processes and sustained increases in O-GlcNAcylation have been linked to the cardiovascular complications associated with diabetes. Conversely, recent studies have shown that acute activation of O-GlcNAc levels protects against hypoxic and ischemic stress. However, despite our increasing appreciation for the significance of O-GlcNAcylation in mediating the response of cardiomyocytes to acute and chronic stress, little is known regarding the fundamental role of O-GlcNAcylation in regulating normal cardiomyocyte function. Therefore, based on our preliminary studies the goal of this proposal is to test the following hypotheses: 1) In the normal heart protein O-GlcNAcylation contributes to regulation of cardiomyocyte gene expression, metabolism and autophagy and 2) Dysregulation in O-GlcNAc synthesis and degradation contribute to the adverse effects of diabetes on the heart including altered gene expression, metabolic dysfunction and impaired autophagic response. Thus the aims of this proposal are to determine the role of O-GlcNAcylation in: 1) The regulation of cardiomyocyte gene expression and e how this is altered in response to type-2 diabetes and identify O-GlcNAc modified cardiomyocyte proteins that are susceptible to acute and chronic changes in O-GlcNAc levels; 2) Acute and chronic regulation of cardiac metabolism and identify the metabolic factors involved in regulating O- GlcNAc turnover in hearts from normal and type-2 diabetic mice; 3) Mediating the balance between cardiomyocyte autophagy and apoptosis in normal and type-2 diabetic cardiomyocytes. The successful completion of the studies outlined in this proposal will significantly enhance our understanding of the impact of this novel metabolically mediated signaling pathway on the cardiomyocyte function under normal conditions as well as how dysregulation in protein O- GlcNAcylation contributes to adverse effects of diabetes on the heart. Consequently, the outcome of this application will be novel mechanistic insights regarding the influence of protein O-GlcNAcylation on myocardial physiology and pathophysiology, potentially identifying novel therapeutic targets.